Insulin resistance is a co-morbidity of obesity and is a risk factor for the development of type-2 diabetes and cardiovascular disease. We, and others, have demonstrated that there is a relationship between accumulated muscle lipids and insulin resistance. Therefore, the focus of this project is two fold: (1) to investigate the mechanism that causes lipids to accumulate in muscle of obese individuals and (2) to investigate the mechanism that links muscle lipids to disregulation of insulin signal transduction. Our hypothesis is that lipids accumulate (e.g. fatty acyl CoAs, diacylglycerols and/or ceramide) in muscle of insulin resistant individuals because of increased uptake of fatty acids across the cell membrane. The accumulated lipids then activate PKC, which leads to the phosphorylation and inactivation of insulin receptors and IRS-1, and insulin resistance. In specific aim 1 we will continue to investigate whether fatty acid transport and expression of fatty acid transporters are altered in muscle of obese individuals. We will use in vivo studies (e.g. hyperinsulinemic clamp) with muscle biopsies or in vitro experiments with the incubated muscle fiber strip preparation, followed by preparation of giant membrane vesicles, to determine if there are alterations in the membrane distribution of fatty acid transporters with obesity (specific aim 2). In specific aim 3 we plan to use cultured muscle myotubes from lean and obese patients to investigate whether manipulating fatty acid transport will alter accumulation of intracellular lipid and insulin signaling. To investigate the link between lipids and insulin signal transduction, we will use our incubated muscle strip preparation to acutely alter the accumulation of muscle lipids and determine if there are accompanying changes in activation of PKC and phosphorylation of insulin receptors and IRS-1 that are consistent with our hypothesis (specific aim 4). The results of the proposed studies should provide valuable information about the causes and possible treatments of insulin resistance and diabetes.